Insecticidal activity of Thymus pallescens de Noë and Cymbogon citratus essential oils against Sitophilus zeamais and Tribolium castaneum

The thrust of the study was to determine the chemical composition of the essential oils extracted from Thymus pallescens de Noé and Cymbogon citratus Stapf. as well as to evaluate their efficacy in controlling Sitophilus zeamais Motschulsky and Tribolium castaneum (Herbst) in either single or combined populations. Carvacrol (56.04%) and geraniol (20.86%) were identified as the major constituents of T. pallescens and C. citratus respectively. The tested essential oils showed pronounced insecticidal activity against the pest species in relation with the applied doses. T. pallescens EO had the highest efficacy and S. zeamais was found to be more susceptible to both individual and combined treatments. With reference to the contact and fumigation assessments, T. pallescens EO effectuated corrected mortality rates ranging from 42.5–100% to 25–100% in S. zeamais with corresponding lethal concentration (LC50) values of 17.7 µl/ml and 15µL/L air respectively. Whereas, the T. pallescens EO exhibited corrected mortality rates of 42.5–100% and 20–100% with corresponding LC50 values of 18.1 µl/ml and 15.5 µL/L air against T. castaneum in contact and fumigation assessments, respectively. The corrected mortality rates increased for both insect species when using combination treatments, with significant increases in the LC50 values, ranging from 8.59 to 49.9% for both pest species. Analysis of energy biomarkers in the treated insects indicate significantly increased protein and carbohydrate contents and decreased lipids levels. The study therefore demonstrated the bio-insecticidal toxicity of the EOs from T. pallescens and C. citratus against two important maize post-harvest pests, concurrently revealing significant positive and negative insecticidal activity gradients in relation to single or combined populations.

The maize weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) is considered as one of the most serious and damaging pests identified in stored corn worldwide.It affects the quantity and quality of the maize grains thus causing severe deterioration of the seed germination potential due to its ability to penetrate into the grain mass 4,7 .S. zeamais is a polyphagous pest species, which accounts for approximately 10-30% yield losses of the annual stored maize grains 8 .
Moreover, maize damaged by S. zeamais activity becomes highly susceptible to other saprophagous species especially Tribolium castaneum (Coleoptera: Tenebrionidae).In turn, infestation with T. castaneum is synonymous with increase in temperature and moisture of stored grains generating an environment that promotes fungal propagation leading to further grain degradation and deterioration 9 .Furthermore, co-existence of different species brings about interspecific competition which results in dramatic losses of the stored grain 10,11 .
To control insect pests during grain storage, chemical synthetic insecticides are frequently used since they can rapidly decimate dense insect populations.However, the indiscriminate use of these chemical compounds results in several adverse environmental impacts such as pest resistance, environmental pollution, disruption of ecological balance and destruction of non-target pollinator insects like bees 12,13 .
Indeed, resistance to several insecticides, such as malathion, pirimiphos-methyl, fenitrothion, and phosphine, has been reported in many storage insect-pests, including S. zeamais and T. castaneum 14,15 .Due to these adverse effects of synthetic chemical pesticides, biopesticides have been explored as a potential alternative 16,17 .In fact, natural insecticides have the advantage of low toxicity for humans and increased biodegradability 18 .
S. zeamais and T. castaneum have been found to be sensitive to various EOs and their active constituents 30,31 .Nonetheless, there is a scarcity of experimental work designed on the effects of EOs on simultaneous infestation of S. zeamais and T. castaneum.Therefore, the aim of this study was to determine the chemical compositions of T. pallescens and C. citratus EOs and to evaluate their insecticidal activity on 2 maize post-harvest pests, S. zeamais and T. castaneum, in single and combined populations.Consequently, we hypothesize that these EOs could negatively influence the physiological responses of the investigated pests.

Plant material collection and air curing
The aerial plant parts of T. pallescens and C. citratus were collected during the flowering season from various localities of Mascara and Algiers, North Algeria.All samples were partially dried for 15 days at room temperature (25 ± 3 °C).

Insect rearing
The study focused on two Coleopteran species: S. zeamais and T. castaneum, which were sampled from infested seed maize from farmers' maize stocks.The insects were reared separately in plastic jars (60 cm × 40 cm × 12 cm) containing a mixture of 1 kg commercial maize and commercial flour according to the proportion of 9/1 (w/w), respectively.The trays were maintained at 26 ± 3 °C and, 70 ± 10% relative humidity (RH), under a 12:12 h (L;D) photoperiod.The adult insects used in all toxicity and biochemical studies were 2-week post-emergence insects of mixed sex 13 .

Essential oil extraction and GC-MS analysis
All EOs were obtained from the aerial parts of T. pallescens and C. citratus by hydrodistillation process for 3 h, using a Clevenger-type apparatus.Initially, a quantity of 100 g of plant material was added to 800 ml of distilled water in a 2-L flask.The set was placed in a balloon heater attached to a refrigerator to ensure condensation of EOs.The yields of EOs were expressed in g relative to 100 g of dry vegetable matter 32 .The T. pallescens leaves gave the highest yield of Eos, ranging from 1.8 to 2.1 g/100 g.The yield of C. citratus ranged from 0.8 to 1.1 g/100 g (mean value for four months).The extracted EOs were dried over anhydrous sodium sulfate and stored at 4°C until use.
Chemical analyses were performed using a system comprised of a 6890 gas chromatography (GC) apparatus using a VF WAX and HP-5MS capillary column (60 m × 0.25 mm × 0.5 µm film thickness) coupled with a Hewlett-Packard computerized 5973A mass spectrometry (MS) apparatus.All EOs were diluted 1/10 (v/v) in hexane, and 1 µL was injected by splitting at a ratio of 1:25.The GC column was a 30m (60m × 0.25mm × 0.5µm film thickness) using a VF WAX and HP-5 capillary column.The GC conditions were as follows: injector temperature, 250 °C; column temperature, isothermal at 60 °C and held for 6 min, then programmed to 250 °C at 6 °C/min and held at this temperature for 2 min; ion source temperature, 250 °C; and detector temperature, 320 °C.Helium was used as the carrier gas at a rate of 0.5 mL/min.The mass range varied from m/z 30 to 350 amu (atomic mass units).The EO components were identified by comparing their retention indices and mass spectra results against those for authentic samples and comparisons of the linear retention indices against a series of n-hydrocarbons.Computer matching was performed against both commercial (NIST 98 and ADAMS) and home-made library mass spectra, based on the analysis of pure substances and the components of known oils derived from data in the MS literature 33 .

Insecticidal activity
The insecticidal activity of the EOs was evaluated against two pest species using two approaches: a separate treatment (each species was treated individually) and a combined treatment (the two species in coexistence).All bioassays were performed under controlled conditions (26 ± 1 °C, 65 ± 5% RH, and a 12:12 h L:D photoperiod).

Contact toxicity
Contact toxicity assays were conducted in accordance with a previously described protocol 34 .Each EO was dissolved in acetone to prepare a serial dilution.Preliminary tests were run to determine the appropriate concentrations that can cause mortality rates ranging from 20 to 80% mortality.For both species, concentrations of 1, 5, 10, 20, 30, 40, 50, 100, 200, 400 µL/mL of each EO were tested.Guided by this assessment the following concentrations were selected for the study: 10, 20, 30, 40, and 50µL/mL.The EOs were applied on the filter paper discs (9 cm diameter) using 2 mL for each trial.Acetone was allowed to evaporate for 10 min prior to the introduction of pest species.Twenty unsexed adults of S. zeamais and T. castaneum were introduced, either separately or together, into Petri dishes containing treated filter paper.The control was maintained under the same conditions without EOs.Four replicates were performed for each treatment.The mortality rates were recorded 72 h after treatment and corrected using Abbott's formula (1925).The insects were considered dead when no movement was recorded.Half-maximal lethal concentration (LC 50 ) values were assessed using probit analysis.

Fumigant toxicity
The fumigant toxicity of EOs was evaluated against S. zeamais and T. castaneum adults in accordance with a previously described technique 13 .Glass jars (1 L) were used as fumigation chambers.As for contact toxicity assay, preliminary tests were conducted in order to determine the concentrations that can effectuate 20 to 80% mortality rates in each of the insect populations.The tested concentrations were 1, 5, 10, 20, 30, 40, 50, 100, 200, 400 µL/L air.Subsequently the following concentrations of EOs were selected and used in the study: 10, 20, 30, 40, and 50 µL/L air.Filter papers (Whatman No. 1) were cut into 9 mm diameter pieces and sprayed with EOs then strongly affixed onto the undersides of the screw caps of the jars.The insides of the jars were brushed with Vaseline to prevent direct contact between the insects and the EO.Caps containing the treated filter paper were tightly screwed onto jars containing twenty adults of S. zeamais and T. castaneum either individually or together.The cover was well-sealed with parafilm.Control insects were maintained under the same conditions with affixed filter paper without EOs.The treatments were replicated four times.Mortality rates were recorded after 72 h of treatment and corrected using Abbott's formula (1925).The LC 50 values were assessed by probit analysis.

Effect of EOs on energy biomarkers, including proteins, carbohydrates, and lipids
To determine proteins, lipids, and total carbohydrate contents, adult insects were treated with three concentrations (30, 40, and 50 µL/mL) of T. pallescens and C. citratus EOs.The control insects used in these experiments were insects that suffered natural deaths.Four replicates were performed for each analysis.

Effect of EOs on proteins
A technique described by Bradford 35 was used for the extraction and quantification of protein reserves.Coomassie Brilliant Blue G-250 (100 mg) was dissolved in 50 mL 95% ethanol (Prolabo; 96% Purity), and 100 mL 85% (w/v) phosphoric acid (Sigma) was added.The resulting solution was diluted to a final volume of 1 L.After crushing the individual insects in 400 µL of the Tris-HCl (20mM) (Sigma; Purity:99.5%)solution, the samples were incubated at 4 °C for 30 min to allow the proteins time to dissolve.An aliquot of 0.1 mL was transferred to a 12 × 100-mm test tube, 5 mL of Bradford reactive was added to the test tube and the contents were mixed by vortexing.The protein concentration was determined by spectrophotometry at 595 nm.The protein concentrations of each sample were determined against a standard curve constructed using 125, 250, 500, 1000 and 2000 µg bovine immunoglobulin G (IgG) dissolved in the same buffer as the samples.Before reading, the plates were gently stirred for 5 s to separate the protein aggregates.

Effect of EOs on carbohydrates
For the extraction and quantification of total carbohydrates, previously described methods were used 36,37 .Insects were ground in 400 µL of sodium sulfate (Prolabo) for 2 min, followed by the addition of 2 mL methanol (Prolabo; 99.8 purity).The tubes containing the homogenate were then centrifuged at 4 °C for 4 min at 2000 × g for 2 min.An aliquot of 100 µL was transferred to a new 12 × 75-mm tube, and 2 mL anthorone reactive was added followed by incubation in a 95 °C water bath for 17 min.The tubes were then placed in an ice bath for 10 min and the optical density at 625 nm was measured.For carbohydrates, a calibration standard curve was generated using a standard glucose solution (1 g/L).The blank was a 0.5 mg/mL glucose solution (5 mg of glucose in 10 mL of distilled water).A series of dilutions were performed to obtain the following glucose concentrations: 10, 20, 40, 60, 80, 100, and 200 µg/mL.

Effect of EOs on lipids
Lipid content was determined using methods described by Van Handel 38 and Plaistow et al. 39 .The insects were crushed in 400 µL chloroform/methanol (1:1, v:v) solution.The supernatant was transferred to a clean tube (16 × 100 mm), which was retained in a water bath at 95 °C, placed inside a fume cupboard to allow the remaining solvent to evaporate.Then, 200 µL of concentrated (95%) sulfuric acid was added, and the solvent was allowed to evaporate at 90 °C for approximately 10 min.The sample was removed from the heating bath, allowed to cool, and 5 mL vanillin-phosphoric acid reagent (85%) was added.The samples were vortexed and then exposed to

Insecticidal activity
The tested EOs exhibited strong insecticidal activity against S. zeamais and T. castaneum adults.A behavioral change was also noticed in both treated insect pests, as indicated by the observation of increased aggregation and couplings between males and females.
The results of the contact toxicity trial expressed as LC 50 of S. zeamais and T. castaneum adults, when tested separately or in combination are shown in Table 3

Fumigant toxicity
The CM% values determined from the fumigant toxicity test clearly indicated that both populations of insect pest species were greatly affected following their exposure to the tested EOs (Table 4).The intensity of CM% varied according to the target pest (F = 7.9; P = 0.006*), the type of EO (F = 110.7;P = 0.000*), the applied concentration (F = 204.9;P = 0.000*), and whether the species were alone or combined (F = 8.7; P = 0.04*).
Interestingly, the results of fumigant toxicity evaluation showed a pronounced increase in CM% compared with those of contact toxicity test.According to the GLM analysis, the population of S. zeamais appeared to have higher sensitivity to both EOs as compared to T. castaneum.The treatment of both separate and combined populations with T. pallescens EO showed higher CM% especially on S. zeamais which was more susceptible than T. castaneum.
Moreover, both pest species were more susceptible when treated alone than when treated together for both studied EOs.In the separate population, T. pallescens EO was found to be more active, with LC 50 values of 15 and 15.5 µL/L air against S. zeamais and T. castaneum, respectively (  6 and 7. Protein content values were significantly affected by the type (F = 98.54;P = 0.000**) and the concentration of the used EO (F = 20.09;P = 0.000*), the pest species (F = 7.08; P = 0.000*), and the toxicity test type (F = 22.28; P = 0.000*).In contact toxicity, S. zeamais and T. castaneum treated with both EOs showed protein levels of 12.57-15.81µg/mg and 12.12-15.71µg/mg, respectively.Whereas, in fumigant toxicity S. zeamais and T. castaneum treated with both EOs presented protein levels of 15.54-22.86µg/mg and 11.55-18.42µg/mg, respectively.Overall, all tested EO concentrations reduced the protein content values in both pest insect species when compared to the control.Significant decreases in protein contents (18.47The carbohydrate contents in S. zeamais and T. castaneum were significantly affected by treatment with T. pallescens and C. citratus EOs (F = 137.8;P = 0.0000*) but were not affected by pest species (F = 0.31; P = 0.56), the type of toxicity test (F = 0.11; P = 0.1), or the EO concentrations (F = 0.3; P = 0.73).In contact toxicity assay, the carbohydrate levels of S. zeamais and T. castaneum treated with both EOs were 1.09-1.51and 1.0-1.43µg/ mg, respectively.The values recorded during fumigation were 1.29-1.68and 1.01-1.73µg/mg, respectively.These results were significantly lower than control levels.Carbohydrates contents were significantly decreased     Lipid content values were significantly affected by the type (F = 102.1;P = 0.0000*) and concentration of EO (F = 36.1;P = 0.000*), the pest species (F = 27.5;P = 0.009*), and the type of toxicity test (F = 23; P = 0.000*).In contact toxicity, the highest levels of lipids were recorded in S. zeamais treated with T. pallescens (1.60-2.59µg/ mg) and C. citratus (1.52-2.02µg/mg ) EOs. While, in fumigant toxicity the highest levels of lipids were recorded in S. zeamais treated with T. pallescens (1.44-2.95µg/mg) and C. citratus (1.20-2.39µg/mg ) EOs.This response was more pronounced with T. pallescens EO than C. citratus EO.The obtained results indicated a significant increase in the lipids contents of S. zeamais and T. castaneum treated with both EOs, with values ranging from 3.56 to 37.68% and from 2.8 to 52.81%, respectively.

Discussion
In the conducted investigations, the chemical composition as well as insecticidal activity of T. pallescens and C. citratus EOs against S. zeamais and T. castaneum adults were assessed in separate and combined populations.According to the results, the major components of T. pallescens were identified as carvacrol (56.64%), p-cymene (16.36%), and thymol (8.71%).Our previous study revealed that carvacrol, thymol, γ-terpinene, and p-cymene were the major components of T. pallescens EO with percentage yields of 54.09, 16.24, and 8.47%, respectively, which is in quite agreement to the obtained results in this study 40 .The EO of the same plant (T.pallescens) has been reported to have similar composition 41 .Regarding C. citratus EO, geraniol (20.86%), limonene (10.50%), and camphene (7.80%) were found to be the major compounds, which is in accordance with previous findings 40,42 .However, the chemical composition of the C. citratus EO was different to that reported by Kumar et al. 43 and Brügger et al. 44 where neral, citral, 1,8-cineole, and geranyl acetate was detected at higher concentrations.These differences are probably due to the crop season and country origin 45 .
The tested EOs in our study are likely rich sources of these components.Benchabane et al. 41 have studied the insecticidal activity of T. pallescens EO and reported that thymol and carvacrol were the components most responsible for this bioactivity.In this study, geraniol (20.86%), limonene (10.50%), and camphene (7.80%) were found to be the most abundant components in C. citratus EO.These compounds have also been reported to have insecticidal activities against a variety of pest insect species 50,52,53 .
Zhang et al. 63 found that EO extracted from the leaves of Chamaecyparis obtusa demonstrated contact and Fumigant toxicity against T.castaneum adults, with respective LC 50 values was 52.54 μg/adult of 7.09 μg/L air.In previous studies, it had been reported that the EO of Artemisia herba alba Asso, Juniperus phoenicea L and Rosmarinus officinalis exerted significant fumigant toxicity against T. castanum (LD 50 = 12.4 μg/adult) 64 .LC 50 values of EOs isolated from Curcuma aromatica salisb showed more contact (at 24 h, LC 50 = 10.40 mg/cm2) and fumigant (at 24 h, LC 50 = 18.18 mg/L air) toxicity against T. castaneum 65 .The contact toxicity studied with Cinnamomum glanduliferum (Wall.)leaf EO showed contact effect against T. castaneum, indicating a low LD 50 / LC 50 value (12.13 μg/adult and 104.67 μg/cm2, respectively) 66 .The fumigant studies with C. austroindica and C. aromatica EOs showed higher toxicity against T. castaneum at the exposure time of 24 h (LC 50= 35.65 & 29.10 μL/L), respectively 67 .Pervious study reported better contact activity for Thymus capitatus and Origanum compactum against T. castaneum.In the contact toxicity, LC 50 values were 0.58 and 0.35 μL/cm2 after 24 h of exposure, respectively 68 .
The toxicity of T. pallescens and C. citratus EOs against two stored-product pests, S. zeamais and T. castaneum, were determined.The CM% results were found to vary with the type and concentration of the tested EOs and the insect species.Thymus pallescens EO demonstrated high toxicity compared to C. citratus EO.Adults of S. zeamais were more susceptible to both plant EOs than T. castaneum.The different responses observed between the two insect species could be attributed to morphological and behavioral differences.It was found that the insecticidal activity of both EOs was highly influenced by the tested toxicity method.Our results are in accordance with previous findings regarding insecticidal activities of various EOs and their major components against S. zeamais and T. castaneum 34,46,58,69,70 .
Furthermore, the obtained results indicated that LC 50 values increased in the combined populations compared with the separate populations.The observed increase in LC 50 values for both pest insects could be due to the increase in the total number of individuals.In addition, the synchronous presence of multiple primary pest species in a mixed-species population can affect control measures, particularly for targeted species 11  www.nature.com/scientificreports/ In fact, EO toxicity can be attributed to different mechanisms of action against the physiological processes in insect pests.Previous studies have shown that natural compounds can cause symptoms associated with neurotoxic activity, such as hyperactivity, seizures, and tremors, often followed by paralysis and death 71 .EOs and their major components also reported to result in the strong inhibition of AChE in congeneric insect pest species, such as S. oryzae 13 .This could explain the toxicity effects of T. pallescens and C. citratus EOs against S. zeamais and T. castaneum.The bioactive properties of EOs can vary depending on the molecules contained in the EOs, the insect species, the toxicity test used, and the dose used 72 .
Plant-derived EOs affect insect metabolism and development through various biochemical and physiological processes 26,73 .In this study, significant decreases in the levels of proteins and carbohydrates, and a significant increase in lipids have been recorded.The decreased protein contents observed for both pest insects may be associated with several factors, including a decrease in protein synthesis or an increase in protein breakdown to detoxify the bioactive molecules of EOs 74,75 .Yazdani et al. 75 obtained similar results using EOs from Thymus vulgaris L. and Origanum vulgare L. against the hemolymph protein of lesser mulberry pyralid Glyphodes pyloalis Walker.
The carbohydrate contents significantly decreased following EO treatments in the tested pest insects.Similar results were obtained by Yazdani et al. 76 in G. pyloalis following treatment with T. vulgaris and O. vulgare EOs and by Tarigan et al. 77 in T. castaneum and Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae) treated with cardamom, cinnamon, and nutmeg EOs.
Insects typically convert carbohydrates into lipids and glycogens 37 , which could explain the decrease in carbohydrate levels and the increased lipid levels observed in the treated insects.The increase in lipid levels can also be explained by the establishment of insect teguments to prevent the harmful effects of EOs.According to Morgan 78 , the external covers of insects consist of an impermeable lipid layer, which typically contains alkanes, methyl branched alkanes, and alkenes.The observed increase in the lipid levels of the insect body may represent the activation of resistance mechanisms to counteract the stress of EOs.The obtained results agree also with the observations of Bouzar Essaïdi et al. 79 , who found that treatments with extracts from Lantana camara L. significantly enhanced lipid contents of the pine processionary moth Thaumetopea pytiocampa.

Conclusion
EOs provide an effective and ecologically sustainable solution for the control of insect pests.Our study demonstrated that T. pallescens and C. citratus EOs were effective against S. zeamais and T. castaneum and could be used as an alternative and "safe" control method for reducing the negative economic impacts associated with these insect pests, as part of an integrated pest management strategy for stored products.Our results also revealed that the synchronized occurrence of S. zeamais and T. castaneum reduced EO efficiencies and, consequently, increased the LC 50 values.
. In the separate population treatments, T. pallescens EO was more active on S. zeamais (LC 50 = 17.7 µL/mL) and T. castaneum adults (LC 50 = 18.1 µL/mL).C. citratus EO was more effective against S. zeamais adults than against T. castaneum with LC 50 values of 46.2 and 57.4 µL/ mL, respectively.The LC 50 values increased significantly under combined population treatment with levels of 8.59-49.9%for both insect pests.Similarly, T. pallescens EO demonstrated the highest insecticidal activity against S. zeamais and T. castaneum in the combined population with LC 50 values of 20.8 and 36.1 µL/mL, respectively.

Table 1 .
to 48.87%) were observed for S. zeamais treated with T. pallescens and C. citratus EOs during contact and fumigant tests.The protein levels in T. castaneum under the same conditions decreased by approximately 9.77 to 33.67%.Chemical constituents of the essential oil from T. pallescens and C. citratus as analyzed by GC-MS.RT retention time; trace.Significant values are in bold.

Table 2 .
Effect of contact toxicity of T. pallescens and C. citratus EOs against S. zeamais and T. castaneum adults in single and combined populations.Values represent the mean of four replicates ± SE (standard errors).Data marked by different letters in a column indicate significant difference at P < 0.05.

Table 3 .
LC 50values of contact toxicity of T. pallescens and C. citratus EOs against S. zeamais and T. castaneum adults in single and combined populations.Values represent the mean of four replicates ± SE (standard errors).LC median lethal concentration, CL confidence intervals.

Table 4 .
Effect of fumigant toxicity of T.pallescens and C.citratus EOs against S. zeamais and T. castaneum adults in single and combined populations.Values represent the mean of four replicates ± SE (standard errors).Data marked by different letters in a column indicate significant difference at P < 0.05.

Table 5 .
LC 50 values of fumigant toxicity of T. pallescens and C. citratus EOs against S. zeamais and T. castaneum adults in single and combined populations.Values represent the mean of four replicates ± SE (standard errors).LC median lethal concentration, CL confidence intervals.

Table 7 .
Effect of fumigant toxicity of T. pallescens and C. citratus EOs on protein, lipids, and carbohydrates quantities in S. zeamais and T. castaneum adults.Values represent the mean of four replicates ± SE (standard errors).Data marked by different letters in a column indicate significant difference at P < 0.05.